Induction of cytotoxic T-lymphocyte responses

ABSTRACT

Methods and compositions useful for inducing a cytotoxic T lymphocyte response (CTL) in a human or domesticated or agriculturally important animal. The method includes the steps of providing the antigen to which the CTL response is desired and providing an antigen formulation which comprises, consists, or consists essentially of two or more of a stabilizing detergent, a micelle-forming agent, and an oil. This antigen formulation is preferably lacking in an immunostimulating peptide component, or has sufficiently low levels of such a component that the desired CTL response is not diminished. This formulation is provided as a stable oil-in-water emulsion.

BACKGROUND OF THE INVENTION

This application is a continuation-in-part of pending U.S. Ser. No.08/919,787 filed Jul. 24, 1992 which is a continuation-in-part of U.S.Ser. No. 07/735,069, filed Jul. 25, 1991, entitled "Induction ofCytotoxic T-Lymphocyte Responses," by Syamal Raychaudhuri and William H.Rastetter (now abandoned). All of these applications are incorporated byreference in their entirety. This invention relates to methods andcompositions useful for inducing cytotoxic T-cell mediated responses inhumans, and domesticated or agricultural animals.

Cytotoxic T-lymphocytes (CTLs) are believed to be the major host defensemechanism in response to a variety of viral infections and neoplastic orcancerous growth. These cells eliminate infected or transformed cells byrecognizing antigen fragments in association with various molecules(termed class I MHC molecules) on the infected or transformed cells.CTLs may be induced experimentally by cytoplasmic loading of certainsoluble antigens within specific cells. Immunization with the solubleantigen alone is generally insufficient for specific cytotoxicT-lymphocyte induction.

One method by which CTL response may be induced involves the use ofrecombinant engineering techniques to incorporate critical components ofan antigen in question into the genome of a benign infectious agent. Theaim of such a strategy is to generate antigen-specific cytotoxicT-lymphocyte responses to the desired epitope by subjecting the host toa mild, self-limiting infection. Chimeric vectors have been describedusing vaccinia, polio, adeno- and retro-viruses, as well as bacteriasuch as Listeria and BCG. For example, Takahashi et al. 85 Proc. Natl.Acad. Sci., USA 3105, 1988 describe use of recombinant vaccinia virusexpressing the HIV gp160 envelope gene as a potential tool for inductionof cytotoxic T-lymphocytes.

A second method by which a cell mediated response may be inducedinvolves the use of adjuvants. While the art appears replete withdiscussion of the use of adjuvants, it is unclear in such art whethercell mediated immunity was induced and whether such cell mediatedimmunity included a cytotoxic T-lymphocyte response. The following,however, are representative of various publications in this area.

Stover et al., 351 Nature 456, 1991 (not admitted to be prior art to thepresent application) describes a CTL response to β-galactosidase usingrecombinant BCG containing a β-galactosidase gene. No such response wasdetected using incomplete Freund's adjuvant and β-galactosidase.

Mitchell et al., 8 J. Clinical Oncology 856, 1990 (which is not admittedto be prior art to the present invention) describe treatment ofmetatastic melanoma patients with an adjuvant termed "DETOX" andallogeneic melanoma lysates administered five times over a period of sixweeks. In a small portion of the patients an increase in cytolyticT-cells was observed. The authors describe a need to enhance the levelof cytotoxic T-lymphocyte production, and suggest a combined therapy ofadjuvant with Interleukin-2, as well as a pretreatment withcyclophosphamide to diminish the level of tumor specific T-suppressorcells that might exist. DETOX includes detoxified endotoxin(monophosphoryl lipid A) from Salmonella minnesota, cell wall skeletonsof Mycobacterium phlei, squalene oil and emulsifier.

Allison and Gregoriadis, 11 Immunology Today 427, 1990 (which is notadmitted to be prior art to the present invention) note that the onlyadjuvant "authorized for use" in human vaccines is aluminum salts (alum)which does not consistently elicit cell mediated immunity. Allison andGregoriadis state " t!here is, therefore, a need to develop adjuvantswith the efficacy of Freund's complete adjuvant but without its variousside effects such as granulomas." They go on to state that threepossible strategies exist, for example, the use of liposomes; the use ofadjuvants, termed immunostimulating complexes (ISCOMs, which includesaponin or Quil A (a triterpenoid with two carbohydrate chains),cholesterol, and phosphatidyl choline) which are authorized for use inan influenza vaccine for horses (Morein et al., Immunological Adjuvantsand Vaccines, Plenum Press, 153); and the use of an emulsion (SAF) ofsqualene or Squalane (with or without a pluronic agent) and muramyldipeptide (MDP). SAF is said to elicit a cell mediated immunity in mice,although it "has long been thought that subunit antigens cannot elicitcytotoxic T-cell (CTL) responses."

Takahashi et al., 344 Nature 873, 1990, describe class II restrictedhelper and cytotoxic T-lymphocyte induction by use of ISCOMs with asingle subcutaneous immunization in mice. They state that Freund'sadjuvant, incomplete Freund's adjuvant, and phosphate buffered salinedid not induce cytotoxic T-lymphocyte activity against the targets inwhich they were interested. They state that, in contrast to results withother forms of exogenous soluble protein antigen, they have shown thatit is possible to prime antigen specific MHC class I restricted CD8⁺ CD4CTL by immunization with exogenous intact protein using ISCOMs. Theyalso state that the experiments described suggest that it may bepossible to elicit human CTL by using ISCOMs containing HIV proteins,and that ISCOM-based vaccines may achieve the long sought goal ofinduction of both CTL and antibodies by a purified protein.

Byars and Allison, 5 Vaccines 223, 1987 describe use of SAF-1 whichincludes TWEEN 80, PLURONIC L121, and squalene or Squalane, with orwithout muramyl dipeptide, and suggest that their data indicate that theformulation with muramyl dipeptide will be useful for human andveterinary vaccines. Booster shots of the adjuvant were provided withoutthe muramyl dipeptide. The muramyl dipeptide is said to increaseantibody production significantly over use of the adjuvant withoutmuramyl dipeptide. Cell mediated immunity was measured as delayed typehypersensitivity by skin tests to determine T-helper cell induction.Such hypersensitivity was stronger and more sustained when muramyldipeptide was provided in the adjuvant. Similar adjuvants are describedby Allison et al., U.S. Pat. No. 4,770,874 (where it is stated that thecombination of muramyl dipeptide and pluronic polyol is essential toelicit a powerful cell mediated and humoral response against eggalbumin); Allison et al., U.S. Pat. No. 4,772,466; Murphy-Corb et al.,246 Science 1293, 1989 (where it is stated that the use of combinedadjuvants with muramyl dipeptide might enhance induction of both humoraland cellular arms of the immune response); Allison and Byars, 87Vaccines 56, 1987 (where it is stated that cell mediated immunity iselicited by SAF (with muramyl dipeptide) as shown by delayed typehypersensitivity, by proliferative responses of T-cells to antigen, byproduction of Interleukin-2, and by specific genetically restrictedlysis of target cells bearing the immunizing antigen); Allison andByars, Immunopharmacology of Infectious Diseases: Vaccine Adjuvants andModulators of Non-Specific Resistance 191-201, 1987; Morgan et al., 29J. Medical Virology 74, 1989; Kenney et al., 121 J. ImmunologicalMethods 157, 1989; Allison and Byars, 95 J. Immunological Methods 157,1986 (where aluminum salts and mineral oil emulsions were shown toincrease antibody formation, but not cell mediated immunity; and muramyldipeptide formulations were shown to elicit cell mediated immunity);Byars et al., 8 Vaccine 49, 1990 (not admitted to be prior art to thepresent application, where it is stated that their adjuvant formulationmarkedly increases humoral responses, and to a lesser degree enhancescell mediated reactions to influenzae haemagglutinin antigen); Allisonand Byars, 28 Molecular Immunology 279, 1991 (not admitted to be priorart to the present application; which states that the function of themuramyl dipeptide is to induce expression of cytokines and increaseexpression of major histocompatibility (MHC) genes; and that betterantibody and cellular responses were obtained than with other adjuvants,and that it is hoped to ascertain whether similar strategies areefficacious in humans); Allison and Byars, Technology Advances inVaccine Development 401, 1988 (which describes cell mediated immunityusing SAF); Epstein et al., 4 Advance Drug Delivery Reviews 223, 1990(which provides an overview of various adjuvants used in preparation ofvaccines); Allison and Byars, 95 J. Immunological Methods 157, 1986(which states that the addition of the muramyl dipeptide to the adjuvantmarkedly augments cell mediated responses to a variety of antigens,including monoclonal immunoglobulins and virus antigens); and Morgan etal., 29 J. Medical Virology 74, 1989 (which describes use of SAF-1 forpreparation of a vaccine for Epstein-Barr virus).

Kwak et al., Idiotype Networks in Biology and Medicine, Elsevier SciencePublishers, p. 163, 1990 (not admitted to be prior art to the presentapplication) describe use of SAF without muramyl dipeptide as anadjuvant for a B-cell lymphoma idiotype in a human. Specifically, anemulsion of Pluronic L121, Squalane, and 0.4% TWEEN-80 in phosphatebuffered saline was administered with the idiotype. They state that "a!ddition of an adjuvant should further augment" . . . humoralresponses, and may facilitate induction of cellular responses as well.

Other immunological preparations include liposomes (Allison et al., U.S.Pat Nos. 4,053,585, and 4,117,113); cyclic peptides (Dreesman et al.,U.S. Pat. No. 4,778,784); Freunds Complete Adjuvant (Asherson et al., 22Immunology 465, 1972; Berman et al., 2 International J. Cancer 539,1967; Allison, 18 Immunopotentiation 73, 1973; and Allison, Non-SpecificFactors Influencing Host Resistance 247, 1973); ISCOMs (Letvin et al.,87 Vaccines 209, 1987); adjuvants containing non-ionic block polymeragents formed with mineral oil, a surface active agent and TWEEN 80(Hunter and Bennett, 133 J. Immunology 3167, 1984; and Hunter et al.,127 J. Immunology 1244, 1981); adjuvants composed of mineral oil andemulsifying agent with or without killed mycobacteria (Sanchez-Pescadoret al., 141 J. Immunology 1720, 1988); and other adjuvants such as alipophilic derivative of muramyl tripeptide, and a muramyl dipeptidecovalently conjugated to recombinant protein (id.).

SUMMARY OF THE INVENTION

Applicant has discovered a safe and advantageous method and compositionsby which CTL responses may be induced in humans and domesticated oragriculturally important animals. The method involves the use of anantigen formulation which has little or no toxicity to animals, andlacks an immunostimulating peptide, (e.g., muramyl dipeptide) thepresence of which would decrease the desired cellular response. Inaddition, the methodology is simple to use and does not requireextensive in vivo work to alter existing cells by recombinant DNAtechniques to make them more antigenic. This discovery is surprisingsince it was unexpected that such a CTL response could be induced by useof such an antigen formulation lacking immunostimulating peptides ortheir equivalent. Applicant's findings allow the use of such antigenformulations in a broad spectrum of disease states, or as a prophylacticagent. For example, such antigen formulation administration can be usedfor the treatment of viral diseases in which a CTL response isimportant, for example, in the treatment of HIV infection or influenza;it can also be extended to use in treatment of bacterial infections,cancer, parasitic infections, and the like. As a prophylactic agent, theantigen formulation combined with a suitable antigen is useful inprevention of infection by viruses responsible for the aforementionedviral diseases, particularly the prophylaxis of HIV infection, and alsofor prophylaxis of patients at risk of cancer, for example, afterresection of a primary tumor.

Thus, in a first aspect the invention features a method for inducing aCTL response in a human or domesticated (e.g., a cat or dog) oragriculturally important animal (e.g., a horse, cow or pig) to anantigen other than B-cell lymphoma antigen or egg albumin. The methodincludes the steps of providing the antigen to which the CTL response isdesired, and providing a non-toxic antigen formulation which comprises,consists, or consists essentially of, a stabilizing detergent, amicelle-forming agent, and a biodegradable and biocompatible oil. Thisantigen formulation preferably lacks any immunostimulating peptidecomponent, or has sufficiently low levels of such a component that thedesired cellular response is not diminished. This formulation ispreferably provided as a stable oil-in-water emulsion. That is, each ofthe various components are chosen such that the emulsion will remain inan emulsion state for a period of at least one month, and preferably formore than one year, without phase separation. In the method the antigenand antigen formulation are mixed together to form a mixture (preferablyby microfluidization), and that mixture administered to the animal in anamount sufficient to induce CTL response in the animal. Suchadministration is required only once.

By "stabilizing detergent" is meant a detergent that allows thecomponents of the emulsion to remain as a stable emulsion. Suchdetergents include polysorbate, 80 (TWEEN)(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured byICI Americas, Wilmington, Del.), TWEEN 40, TWEEN 20, TWEEN 60,Zwittergent 3-12, TEEPOL HB7, and SPAN 85. These detergents are usuallyprovided in an amount of approximately 0.05 to 0.5%, preferably at about0.2%.

By "micelle-forming agent" is meant an agent which is able to stabilizethe emulsion formed with the other components such that a micelle-likestructure is formed. Such agents preferably cause some irritation at thesite of injection in order to recruit macrophages to enhance thecellular response. Examples of such agents include polymer surfactantsdescribed by BASF Wyandotte publications, e.g., Schmolka, 54 J. Am. Oil.Chem. Soc. 110, 1977, and Hunter et al., 129 J. Immunol 1244, 1981, bothhereby incorporated by reference, PLURONIC L62LF, L101, and L64,PEG1000, and TETRONIC 1501, 150R1, 701, 901, 1301, and 130R1. Thechemical structures of such agents are well known in the art.Preferably, the agent is chosen to have a hydrophile-lipophile balance(HLB) of between 0 and 2, as defined by Hunter and Bennett, 133 Journalof Immunology 3167, 1984. The agent is preferably provided in an amountbetween 0.5 and 10%, most preferably in an amount between 1.25 and 5%.

The oil is chosen to promote the retention of the antigen inoil-in-water emulsion, i.e., to provide a vehicle for the desiredantigen, and preferably has a melting temperature of less than 65° C.such that emulsion is formed either at room temperature (about 20° C. to25° C.), or once the temperature of the emulsion is brought down to roomtemperature. Examples of such oils include squalene, Squalane, EICOSANE,tetratetracontane, glycerol, and peanut oil or other vegetable oils. Theoil is preferably provided in an amount between 1 and 10%, mostpreferably between 2.5 and 5%. It is important that the oil isbiodegradable and biocompatible so that the body can break down the oilover time, and so that no adverse affects, such as granulomas, areevident upon use of the oil.

It is important in the above formulation that a peptide component,especially a muramyl dipeptide (MDP) be lacking. Such a peptide willinterfere with induction of a CTL response if it provided in an amountgreater than about 20 micrograms per normal human formulationadministration. It is preferred that such peptides be completely absentfrom the antigen formulation, despite their apparent stimulation of thehumoral compartment of the immune system. That is, applicant has foundthat, although such peptides may enhance the humoral response, they aredisadvantageous when a cytotoxic T-lymphocyte response is desired.

In other related aspects, the antigen formulation is formed from onlytwo of the above three components and used with any desired antigen(which term includes proteins, polypeptides, and fragments thereof whichare immunogenic) except egg albumin (or other albumins, e.g., HSA, BSAand ovalbumin), to induce a CTL response in the above animals or humans.

Applicant believes that the above formulations are significantlyadvantageous over prior formulations (including ISCOMs, DETOX, and SAF)for use in humans. Unlike such formulations, the present formulationboth includes a micelle-forming agent, and has no peptides, cell wallskeletons, or bacterial cell components. The present formulation alsoinduces a CTL response which either does not occur with the priorformulations, or is significantly enhanced compared to thoseformulations.

By "non-toxic" is meant that little or no side effect of the antigenformulation is observed in the treated animal or human. Those ofordinary skill in the medical or veterinary arts will recognize thatthis term has a broad meaning. For example, in a substantially healthyanimal or human only slight toxicity may be tolerated, whereas in ahuman suffering from an imminently disease substantially more toxicitymay be tolerated.

In preferred embodiments, the antigen formulation consists essentiallyof two or three of the detergent, agent, and oil; the method consistsessentially of a single administration of the mixture (antigen plusantigen formulation) to the human or the animal; the human or animal isinfected with a virus and suffers one or more symptoms (as generallydefined by medical doctors in the relevant field) of infection from thevirus; and the antigen formulation is non-toxic to the human or animal.

In other preferred embodiments, the antigen is chosen from antigenicportions of the HIV antigens: gp160, gag, pol, Nef, Tat, and Rev; themalaria antigens: CS protein and Sporozoite surface protein 2; theHepatitis B surface antigens: Pre-S1, Pre-S2, HBc Ag, and HBe Ag; theinfluenza antigens: HA, NP and NA; Hepatitis A surface antigens; theHerpes virus antigens: EBV gp340, EBV gp85, HSV gB, HSV gD, HSV gH, HSVearly protein product, human papillomavirus antigens (e.g., HPVantigens, such as L1, E4, E6, E7 antigens, in particular the E6 and E7antigens from HPV16 and 18, the two most common HPV types associatedwith cervical carcinoma, E4 and L1 derived from HPV6 and HPV11, the twomost conunon HPV types associated with condyloma acuminata; the prostatespecific antigen (PSA), cytomegalovirus gB, cytomegalovirus gH, and IEprotein gP72; the respiratory syncytial virus antigens: F protein, Gprotein, and N protein; and the tumor antigens carcinoma CEA, carcinomaassociated mucin, carcinoma P21, carcinoma P53, melanoma MPG, melanomap97, and carcinoma Neu oncogene product, carcinoma p53 gene product, themelanoma antigen called MAGE, and mutated p21 ras protein presented in avariety of malignant tumors.

In related aspect, the invention features a composition comprising,consisting, or consisting essentially of an antigen mixed with anantigen formulation described above, and the antigen is chosen fromthose antigenic portions listed above.

In other related aspects, the invention features methods of treating apatient infected with HIV virus, suffering from malaria, suffering frominfluenza, suffering from hepatitis, suffering from a cancer, infectedwith herpes virus, suffering from cervical cancer, suffering fromcondyloma acuminata (genital warts), or infected with respiratorysyncytial virus, by administering a composition including an appropriateantigen (e.g., selected from those listed above) mixed with one of theabove antigen formulations. These antigens and treatments are onlyexemplary of antigens which may be used in the subject antigenformulations.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

Drawings

FIGS. 1A-1C and 4A-4C are graphical presentations of data comparing CTLinduction by various ovalbumin formulations; E:T represents effector totarget ratio in all Figures.

FIGS. 2A and 2B are graphical presentations of data comparing CTLinduction by various β-galactosidase formulations;

FIG. 3 is a graphical presentation of data comparing CTL induction byovalbumin in a liposome and in an antigen formulation;

FIGS. 5 and 6 are graphical presentations of data showing the effect ofCD4 and CD8 cell depletion on CTL induction;

FIG. 7 is a graphical representation of data showing CTL induction bygp120;

FIG. 8 is a graphical representation of data showing CTL induction by amixture of pluronic and TWEEN and an antigen;

FIG. 9 is a graphical representation of data showing CTL induction witha mixture of squalane and pluronic and an antigen;

FIG. 10 is a graphical representation of data showing CTL induction by amixture of squalane and pluronic and an antigen;

FIG. 11 is a graphical representation of the effect of OVA with variousantigen formulations on CTL response;

FIG. 12 is a graphical representation of the induction of anti-gp120IIIbantibodies in monkeys with various antigen formulations;

FIG. 13 depicts antitumor activity of HOPE2 cells ten days after asingle immunization of soluble E7 protein in adjuvant; and

FIG. 14 depicts antitumor activity of HOPE2 cells at days 10, 19 aftertwo immunizations with soluble E7 protein in adjuvant.

Antigen Formulation

Antigen formulations useful in this invention are generally describedabove. Those of ordinary skill in this art will recognize thatequivalent Formulations are readily prepared and can be expected to haveequivalent properties in induction of a CTL response. Such Formulationsare readily tested for their properties using techniques equivalent tothose described in the examples below.

There follow examples of the invention with the use of an antigenformulation (AF) composed of about 2.5% squalane, 5% pluronic acid, andTWEEN 80 in a phosphate buffered saline. Specifically, an emulsion ofthe AF included: 15 mg squalane, 37.5 mg poloxamer 401 (PLURONIC L121),6 mg polysorbate 80 (TWEEN 80), 0.184 mg potassium chloride, 0.552 mgpotassium phosphate monobasic, 7.36 mg. sodium chloride, 3.3 mg sodiumphosphate dibasic (anhydrous), per 1 ml water, pH 7.4. This emulsion wasmicrofluidized using standard technique (Microfluidics Model M110F) witha back-pressure module at 11-14,000 psi with gradual return toatmosphere pressure, cooling and packing in wet ice.

In other examples, antigen was mixed with the microfluidized squalane(S), pluronic (P) and TWEEN 80 (T) mixture to achieve a finalconcentration of 0.2% TWEEN 80, 1.25% pluronic and 5% squalanerespectively. To determine the sub-components necessary for an antigenspecific immune response induction, Squalane-TWEEN 80, pluronic-TWEEN 80or Squalane-pluronic were prepared at the same concentration as for thethree components mixture. Pluronic, Squalane or TWEEN 80 was alsoprepared individually to determine the effect of individual component onthe CTL induction. Substitutions of TWEEN 20, TWEEN 40 or Zwittergentfor TWEEN 80 were also made to determine the effect of various TWEENderivative on the CTL induction in the ova system. Substitutions ofSqualane in the three component formulation were made with Eicosone orTriacontone and substitution for the co-polymer pluronic in the samethree components formulation were made by PEG 1000, Pleuronic L62LF, andthe Tetronics 1501 and 150R1. As two component formulations, variousanalogs in various combinations were mixed and tested for ova specificCTL induction. They are a mixture of cholesterol--TWEEN 80,Squalane--TWEEN 20, Pristane--TWEEN 80 or olive oil--TWEEN 80. For astabilization study, the microfluidized mixture of Squalane-TWEEN 80 wasmixed with dextrose to a final concentration of 5%. In all cases thecombinations of excipients were mixed in a microfluidizer to made astable emulsion. In some experiments, two components formulations weremixed with various concentration of MDP for CTL and humoral responseinductions. Table 1 describes a comprehensive list of variousformulations used in this study.

                  TABLE 1                                                         ______________________________________                                        Effect of various substitution in three or two component systems                                  percent kill at E:T 100:1                                 ______________________________________                                        Substitution in three component formulations                                  STP                   84                                                      Tween 40 (T)          66                                                      Tween 20 (T)          48                                                      T1501 (P)              0                                                      TI50R1 (P)             0                                                      Pluronic L62LF (P)    47                                                      Eicosane (S)          *                                                       PEG1000 (P)           *                                                       Triacontane (S)       *                                                       Zwittergent (T)       *                                                       Substitution in two component formulations                                    ST                    76                                                      PT                    45                                                      SP                    26                                                      Cholesterol (S) + Tween 80                                                                           0                                                      Squalane + Tween 29 (T)                                                                             65                                                      Pristane (S) + Tween 80                                                                             42                                                      Olive Oil (S) + Tween 80                                                                            69                                                      1 component formulation                                                       Pluronic L121          0                                                      Squalane               0                                                      Tween 80               0                                                      Squalane + Tween 80 + 5% dextrose                                                                   86                                                      ______________________________________                                         * CTL assay is being repeated                                            

Syntex adjuvant formulation (microfluidized; SAFm) was used as anadjuvant control and consists of two parts. Part I consists of phosphatebuffered saline containing a final concentration of 5% Squalane, 1.25%pluronic and 0.2% TWEEN 80 (vehicle or I-SAF). Part II consists ofN-Acetylmuramyl-L-Threonyl-D-Isoglutamine (Thr-MDP), a derivative ofmycobacterium cell wall component. For immunization purposes, antigen ismixed with microfluidized vehicle (part I) to obtain a homogeneousemulsion. MDP is added to made SAFm, and vortexed briefly. The MDPconcentration in the mixture was varied to determine if there was anoptimum concentration for CTL induction. As an adjuvant control, micewere also immunized with soluble antigens mixed with alum according tothe manufacturer's manual (Pierce Chemical, Rockford, Ill.) or withComplete Freund's Adjuvant (CFA).

This antigen formulation is used for induction of cytotoxic T-lymphocyteresponses in mice. Those of ordinary skill in the art will recognizethat such a mouse model is indicative that equivalent experiments ortreatments will similarly induce cytotoxic T-lymphocyte responses inhumans, domesticated, or agricultural animals. The amount of antigenformulation and antigen useful to produce the desired cellular responsemay be determined empirically by standard procedures, well known tothose of ordinary skill in the art, without undue experimentation. Thus,if desired to minimize the side effects of treatment with such a mixturethose of ordinary skill in the art may determine a minimum level of sucha mixture for administration to a human, domesticated, or agriculturalanimal in order to elicit a CTL response, and thereby induce immunity toa desired antigen. In normal use, such a mixture will be injected by anyone of a number of standard procedures, but particularly preferred is anintramuscular injection at a location which will allow the emulsion toremain in a stable form for a period of several days or several weeks.

Methods

The following materials and methods were used in the examples providedbelow unless otherwise noted:

Mice

Female C57BL/6 (H-2^(b)) and BALB/c (H-2^(d)) mice were purchased fromHarlen Sprague (San Diego, Calif.).

Antigens

Ovalbumin (ova, Grade VII; Sigma Chemical Co., St. Louis, Mo.) was usedin the native form. β-galactosidase, (β-gal, Grade VIII; BRL) was usedin the native form and after boiling in 1 M NaOH for 2 min to give analkali digest. Recombinant gp120 was purchased from AmericanBiotechnology.

Tumor Cells and Transfectants

The tumor cells used were the Ia lines EL4 (C57BL/6, H-2^(b) thymoma)and P815 (DBA/2, H-2^(d) mastocytoma). Derivation of the ova-producingEL4 transfectant, EG7-ova, is described previously by Moore et al., 54Cell 777, 1988. The β-gal-producing transfectant, P13.1, was derived byelectroporation of 10⁷ P815 cells in 1 ml of phosphate buffered saline(PBS) with 10 mg of PstI linearized pCH110 (Pharmacia LKB BiotechnologyInc., Piscataway, N.J.) and 1 mg of PvuI linearized pSV2 neo (Southernet al., 1 J. Mol. Appl. Genet. 327, 1982) followed by selection in 400μg/ml of the antibiotic G418. The C3-4 transfectant was derived from theBALB/c hybridoma Igm 662 by transfecting with a plasmid encoding theβ-gal gene fused to the third and fourth exon of IgM heavy chain(Rammensee et al., 30 Immunogenetics 296, 1989). The gp160IIIbexpressing 3T3 fibroblast, 15-12, was provided by Dr. Germain of NIH(Bethesda, Md.). The K^(b) transfected L cell line was provided by Dr.Carbone, Monash University, Australia. The D^(d) and L^(d) transfected Lcell lines were provided by Dr. Ted Hensen, Washington University, St.Louis.

Immunization

Mice were immunized intravenously with a 200 μl suspension of 25×10⁶splenocytes, after a cytoplasmic loading as described by Moore et. al.supra, and Carbone et al., J. Exp. Med. 169:603, 1989). For ova-antigenformulation or β-gal-antigen formulation immunization, 30 μg of eachprotein antigen was injected per mouse in the footpad and the tailbasesubcutaneously. Each injection consists of 67 μl of microfluidizedantigen formulation (made following standard procedures) and 30 μg ofprotein antigen in a final volume of 200 μl. The final volume was madeup with HBSS, see, Whittaker manual (Welkersville, Md.). MDP wasprovided in concentrations between 0 and 300 μg. Where stated, mice wereimmunized with soluble antigens in CFA, or in alum in a total volume of200 μl.

In vitro stimulation of effector populations

Spleen cells (30×10⁶) from normal or immunized mice which had beenprimed at least 14 days earlier were incubated with 1.5×10⁶ EG7-ova(irradiated with 20,000 rad) for ova responses or 1.5×10⁶ C3-4 cells(irradiated with 20,000 rad) for β-gal response in 24 well plates at 37°C. in 7% CO₂ /air. All the tissue cultures were performed in a completemedium consisting of IMDM medium, see, Whittaker Manual (Welkersville,Md.) supplemented with 10% fetal calf serum (FCS), 2mM glutamine,gentamycin and 2×10⁻⁵ M 2-mercaptoethanol. For the in vitro depletionexperiments, in vivo primed or in vitro stimulated spleen cells weretreated with monoclonal antibodies (mAbs) RL.172 (anti-CD4) or mAbs3.168 (anti-CDS) for removal of CD4⁺ or CD8⁺ T cells (Sarmiento et al.,125 J. Immunol. 2665, 1980, and Ceredig et al., 314 Nature 98, 1985).The mAb RL.172 and mAb 3.168 were obtained from Dr. Jonathan Sprent atScripps Clinic and Research Foundation, La Jolla, Calif.

Spleen cells (30×10⁶) from normal or immunized mice which had beenprimed at least 21 days earlier were incubated with 1.5×10⁶ 15-12 cells(treated with 200 ug of mitomycin C for 45 minutes per 10⁸ cells), orwith 500 μg of 18IIIb peptide containing the dominant CTL epitope inBalb/c mice in complete IMDM media (Irvine Scientific, Santa Ana,Calif.) containing 10% pre-screened FCS (ICN Flow; ICN Biochemicals,Inc., Costa Mesa, Calif.), 2 mM glutamine, gentamycin and 2×10⁻⁵ M2-mercaptoethanol. For in vitro stimulation with peptides, spleen cellswere cultured in complete IMDM containing 5% ConA supernatant.

For depletion experiments, in vivo primed or in vitro stimulated spleencells were treated with mAbs RL.172 (anti-CD4) or mAbs 3.168 (anti-CDS)in presence of low tox. rabbit complement (Cederlane Laboratories, Ltd.,Hornby Ontario, Canada) for removal of CD4⁺ or CD8⁺ T cells (22, 23).The mAb RL.172 and mAb 3.168 were a gift from Dr. Jonathan Sprent atScripps Clinic and Research Foundation, La Jolla, Calif.

Cytoxicity Assay

Target cells (1×10⁶) were labeled with 100 μCi ⁵¹ Cr! sodium chromatefor 60 min. For peptide pulsed targets, 50 μl of a 1 mg/ml peptidesolution in HBSS was added during the targets labeling with ⁵¹ Cr. Afterwashing, 104 labeled targets and serial dilutions of effector cells wereincubated in 200 μl of RP10 for 4 h at 37° C. 100 μl of supernatant wascollected and the specific lysis was determined as: Percent specificlysis=100×{(release by CTL-spontaneous release)/(maximalrelease-spontaneous release)}. Spontaneous release in the absence ofcytotoxic T-lymphocyte (CTL) was <25% of maximal release by detergent inall experiments.

Determination of Antibody Responses in Mice and Monkeys.

Each well of 96-well, U bottomed plates (Costar, Cambridge, Mass.) werecoated with 150 ng of ova or gp120 in 50 ul of HBSS and incubatedovernight at 4° C. For the determination of anti-gp120 and anti-ovaantibody responses in mice, plates were blocked with 1% BSA for 1 hr.Serially diluted sera were added in 25 μl volume per well and incubatedfor 2 hrs. Plates were washed and 50 μl of 1:1000 dilution of goatanti-mouse IgG conjugated to HRPO (SBT, Alabama) in 1% BSA were addedper well. After 1 hr of incubation, plates were washed and 100 μl ofsubstrate was added per well. The OD405 was taken after 10 to 15minutes. For the determination of monkey anti-gp120 antibody response,all the steps were the same except both the blocking of plates and thedilution of sera were done in 5% normal goat serum in Hank's balancedsalt solution.

Peptide Synthesis

Synthetic peptides corresponding to amino acid sequences 253-276(Sequence Listing No. 1: EQLESIINFEKLTEWTSSNVMEER; where the standardone letter code is used to represent each amino acid) of ovalbumin (ova253-276), amino acid sequences 84-102 of myelin basic protein (MBP84-102) (Sequence Listing No. 2: DENPVVHFFKNIVTPRTPP), and syntheticpeptides corresponding to amino acid sequences 308-322 (18IIIb sequence)of gp120IIIb, were assembled by solid phase peptide synthesis using anApplied Biosystems 430A synthesizer. Amino acids were coupled viapre-formed symmetric anhydrides with the exception of asparagine,glutamine and arginine which were coupled as hydroxybenzotriazoleesters. Coupling efficiency was monitored by ninhydrin reactionfollowing the method of Kaiser et al. 34 Anal. Biochem. 595, 1970. Thepeptides were released from the support with HF following the "low-high"procedure described by Tam, et al. 21 J. Am. Chem. Soc. 6442, 1983, andthe peptides extracted from the resin with 10% acetic acid. Afterlyophilization, peptides were desalted on a Sephadex G-25 column, andsamples of the peptides then HPLC purified by reverse phasechromatography on a Vydac preparative C-18 column. Purified peptides(98%) were solubilized in HBSS at a final concentration of 10 mg/ml anddiluted to the desired concentration in the complete media.

CNBr Digest

Samples of protein (e.g., β-galactosidase) were treated with 100 foldmolar excess of cyanogen bromide in a solution of 100 mM trifluoroaceticacid. The reaction was allowed to proceed for 18 hours at roomtemperature (about 20° C.) with rotation. Following the prescribedreaction time, the peptide fragments were separated from the reactantusing a SEP-PAK C-18 apparatus (Waters), eluted with 95% acetonitrile,and lyophilized.

Alkaline digest

Protein samples (e.g., β-galactosidase) were treated with 1 N NaOH andboiled for 2 minutes, and the resulting peptide fragments were separatedfrom the reactants using a C-18 SEP-PAK apparatus (Waters), and elutedwith 95% acetonitrile and lyophilized.

EXAMPLE 1 Class I restricted CTL priming

Moore et al., 113 UCLA Symp. Mol. Cell. Biol. 1989 and Carbone andBeyan, 171 J. Exp. Medicine 377, 1990, demonstrate that mice immunizedwith spleen cells loaded cytoplasmically with soluble ova, were primedfor ova specific, class I restricted CTL response. The ova-expressingEL4 transfectant EG7-ova was employed for in vitro stimulation of invivo primed splenic lymphocytes and also used as target for ova specificCTL mediated killing. This study also demonstrated that CD8⁺ effectorsinduced by EG7-ova transfectant or by spleen cells cytoplasmicallyloaded with ova, recognize a determinant mapped by the peptide ova258-276 in the context of H-2K^(b), lyse EG7-ova, and also kill EL4cells coated with ova 258-276. Thus, in order to assess whether anendogenous class I restricted CD8⁺ T cell pathway can be induced by asoluble antigen, the above system was used to determine whether certainantigen formulations can be used to drive soluble antigen into a class Irestricted pathway.

a) ova

C57BL/6 mice were immunized once with various amounts of ova (30 μg--1mg per mouse) with or without an antigen formulation. Mice were injectedsubcutaneously and in the tailbase. Spleen cells were taken from theimmunized mice at least two weeks after the immunizations and in vitrostimulated with the EG7-ova transfectants. An ova concentration as lowas 30 μg was as effective as a 1 mg dose. Therefore, the CTL studieswere routinely performed with spleen cells from 30 μg ova-primed mice.After five days of in vitro culture with EG7-ova, priming was assessedby the presence of ova specific effectors capable of lysing EG7-ova.

Mice injected with soluble ova in HBSS as high as 1 mg, showed noevidence of CTL priming (FIG. 1A). However mice immunized with 30 μg ovain the antigen formulation described above (shown as AF in the figures)showed a significant transfectant specific CTL response (FIG. 1C).Furthermore, the extent of EG7-ova killing by the ova-AF immunizedspleen cells was comparable to that of ova-loaded spleen cells immunizedmice (FIG. 1B).

That the specificity of CTL priming in vivo was antigen specific wasshown by the lack of spleen cells from β-galactosidase immunized mice tomanifest secondary CTL response in vitro when stimulated with EG7-ova.No ova specific CTL induction was observed.

b) β-galactosidase

Similar results were obtained using another soluble protein antigen,β-gal. For assaying β-gal-specific CTL response, the target used wasBALB/c derived β-gal-expressing C3-4 transfectant. Immunization ofBALB/c mice with soluble β-gal gave background CTL response. Therefore,for the determination of specific CTL response, harvesting was postponedfor at least eight weeks before spleen lymphocytes were harvested, andcultured for five days in the presence of irradiated C3-4 transfectants.

FIG. 2B demonstrates that 30 μg of β-galactosidase in AF induced strongspecific CTL response against transfectant. At an effector-to-target(E:T) ratio of 3:1, β-gal-AF immunized mice showed about 80% of specificC3-4 killing. However, only 20% killing of the same target was achievedwith effectors isolated from β-gal in HBSS immunized mice at the sameE:T ratio (FIG. 2A). Since neither EL4 nor P815 expresses class II MHCgene products and the lysis shows syngeneic restriction, these ova andβ-gal specific effectors are class I MHC restricted.

To demonstrate the usefulness of the antigen formulation, mice wereimmunized with soluble ova encapsuled in two types of liposomes, one ofwhich was a pH sensitive liposome. One week later, spleen cells werestimulated in vitro, as described above, and tested against ⁵¹Cr-labeled EG7-ova or EL4. FIG. 3 shows a representative resultdemonstrating that ova in liposome could not prime mice for substantialCTL induction. Similar results were observed when ova was immunized inalum.

EXAMPLE 2 Recognition of epitope by CTL

Carbone and Bevan, supra, demonstrated that CTL induced in C57BL/6 miceby EG7-ova transfectant, and by cytoplasmically ova-loaded splenocytesrecognize EL4 cells coated with the peptide ova 258-276. To determinewhether soluble ovalbumin in AF induces similar CTL responses, spleencells were prepared from immunized mice and stimulated in vitro withEG7-ova. The effectors were tested against EL4 cells coated with thepeptide ova 253-276, or with a control peptide derived from myelin basicprotein (MBP 84-102). The results demonstrate that ova-AF primed CTLwith a similar specificity to those primed by transfectants, or bycytoplasmically loaded ova (FIGS. 1A, 1B and 1C). ova-AF primed effectorcells effectively lysed EG7-ova, and an untransfected EL4 cells coatedwith 50 μg/10⁸ cells of ova peptide, but did not lyse EL4 cells coatedwith 50 μg/10⁸ cells of MBP peptide.

In the β-galactosidase system, Carbone and Bevan, supra, indicated thatβ-gal expressing transfectant and splenocytes cytoplasmically loadedwith soluble β-galactosidase, induced CTL which lysed β-gal expressingtransfectant and nontransfectant P815 cells coated with alkali digestedβ-galactosidase. Soluble β-galactosidase induces CTL having similarspecificity when immunized in AF (FIG. 2).

EXAMPLE 3 CTL effectors are CD8⁺ T cells

That soluble protein antigens in AF induce CD8⁺ effector T cells wasshown as follows. Splenocytes from immunized mice were cultured for fivedays with irradiated transfectants in vitro. Thereafter, cells wereharvested and depleted of CD4⁺ or CD8⁺ T cells by using monoclonalanti-CD4 or anti-CD8 antibodies plus complement. Depleted populationswere then tested against ⁵¹ Cr-EG7-ova in the ova system or ⁵¹ Cr-P13.1in the β-gal system. The data shown in FIG. 4 indicates that, in the ovasystem, depletion of CD8⁺ T cells abrogated cytolytic activity conferredby the whole effector cell population. However, depletion of CD4⁺ T cellpopulation did not have any effect on the lysis of EG7-ova.

Similarly, in the β-gal system, depletion of CD8⁺ T cells abrogated thecytolytic activity of β-gal-antigen formulation immunized spleen cells.

EXAMPLE 4 Soluble ova in AF prime CD8⁺ T cells

To demonstrate that ova-AF primes CD8⁺ T cell populations in vivo, andis critical for in vitro secondary response, CD4⁺ or CD8⁺ populationswere depleted from spleens of ova-AF immunized mice and from naive mice.These treated populations were then stimulated in vitro with EG7-ovaalone, or in a combination of CD4⁺ and CD8⁺ T cells from ova-AFimmunized mice, or in various combination of CD4⁺ or CD8⁺ T cells fromova-AF immunized mice with the CD4⁺ or CD8⁺ cells from naive mice. FIG.5 shows that primed CD8⁺ cells are essential for the manifestation of asecondary CTL response in vitro. These data also indicate that for theeffective secondary CTL response in vitro, CD4⁺ T cells are required.CD4⁺ cells are not needed for priming. Similarly, CD8⁺ T cells wererequired for the manifestation of B-gal specific secondary CTL responsein vitro.

The above examples demonstrate the effect of the antigen formulation onthe induction of class I restricted CTL responses against solubleprotein antigens. The antigen formulation mediated soluble antigeninduced CTL priming, and is similar in activity to that induced bytransfectants and by splenocytes cytoplasmically loaded with soluble ovaor β-gal. In the ovalbumin system, EG7-ova, cytoplasmically loaded ovasplenocytes, and ova-AF induced: (a) class I restricted CD8⁺ CTL; (b)CTL that recognize target sensitized with ova 253-276 synthetic peptide;and (c) long lived CTL after only one immunization. In theβ-galactosidase system, the β-gal-AF induced CTL that recognize β-galexpressing transfectant C3-4, and also the untransfected P815 cellssensitized with alkali digested β-gal. This is analogous to what wasobserved with CTL induced by immunization with spleen cellscytoplasmically loaded with β-galactosidase. The induction ofova-specific CTL by antigen formulation is unique because neither ovaencapsulated in a pH sensitive liposome, nor in alum, could induce CTLpriming in vivo.

These examples indicate that the antigen formulation used above, and itsequivalents, are useful in human therapy and in vaccine development forthe induction of CTL in various cancers and viral diseases.

EXAMPLE 5

This is a specific example to show the use of the above AF on producingclass I restricted CTL priming by soluble gp120 from HIV.

The gp160 IIIB expressing cell line (15-12) was produced in the Balb/cfibroblast-derived 3T3 cell line. It was obtained from Drs. Ron Germainand Jay Berzofsky, National Institute of Health, Bethesda, Md. The gp160expressing cell line was employed for in vitro stimulation of in vivoprimed splenic lymphocytes, and also used as target for gp160 specificCTL induction. Balb/c mice were immunized once with 10 μg of gp160 permouse with or without AF. Mice were injected at footpads and tailbasesubcutaneously. Spleen cells were taken from the immunized mice aftertwo weeks of immunizations and in vitro stimulated with irradiated gp160transfectants. After five days of culture in vitro, priming was assessedby the presence of specific effectors capable of lysing gp160transfectants, and not the untransfected cell lines. The results areshown in FIG. 7, where CTL response is potentiated with AF and gp120.

The following example demonstrates the use of antigen formulations ofthis invention with use of only one or two components. These examplesdemonstrate that CTL-responses can be induced with only two of the abovethree components.

EXAMPLE 6 Determination of critical components necessary for CTLinduction

To determine whether all the above-noted components are necessary forantigen specific CTL induction, mice were immunized with ovalbumin in amicrofluidized formulation of various combinations of two of the threecomponents presented in the AF above. Two component combinations usedwere as follows; Squalane/TWEEN in PBS, Squalane/Pluronic in PBS orPluronic/TWEEN in PBS. Another set of groups were included where micewere immunized with ova formulated in a one component system i.e.,Squalane in PBS, Pluronic in PBS or TWEEN in PBS only. The above threecomponent antigen formulation was modified to exclude one component at atime, constituting PBS in its place.

The above antigen formulations consist of: 0.300 g TWEEN 80 (Aldrich,Wis.), 1.875 g Pluronic L121 (BASF, NJ), and 7.5 g Squalane (Aldrich,Wis.), brought to 50 ml with PBS.

The two-component formulations were:

Squalane/TWEEN: 0.300 g TWEEN 80, and 7.5 g Squalane, brought to 50 mlwith PBS.

Pluronic/TWEEN: 1.875 g Pluronic L121, and 0.300 g TWEEN 80, brought to50 ml with PBS.

Pluronic/Squalane: 1.875 g Pluronic L121, and 7.5 g Squalane, brought to50 ml with PBS.

The samples were then processed through a microfluidizer, model 110T,Microfluidics corp, and bottled and stored at 4° C. until use.

Ovalbumin (Sigma, Mo.) was weighted and brought to a 0.3 mg/ml solutionin HBSS (Whittaker, Supra). The stock 0.3 mg/ml solution was combinedwith the two component formulation in the following amounts: 5 partsOvalbumin 0.3 mg/ml solution, 3.3 parts 2 component formulation, and 1.7parts HBSS.

The formulation was vortexed and kept on ice until injected. Allsolutions were combined just prior to injection.

Each mouse received 200 μl of one formulation containing 30 μl of OVA byinjection in both hind footpads and any remaining solution was injectedsubcutaneously at the tail base. Mice were allowed to rest for two tofour weeks prior to spleen harvest.

Two weeks after immunizations, spleen cells were prepared and in vitrostimulated with irradiated EG7-OVA. After five days of culture, thepresence of OVA specific CTL was measured by testing against ⁵¹Cr-EG7-OVA or ⁵¹ Cr-EL4 in a 4 hour ⁵¹ Cr release assay. The data shownin FIGS. 8-10 demonstrate that Ovalbumin formulated in microfluidizedtwo component system can prime OVA specific CTLs in vivo.

We further evaluated the relative contribution of the individualcomponents for their ability to induce CTL when combined with proteinantigens. For immunization purposes soluble antigen was mixed withmicrofluidized excipients to obtain a stable homogeneous emulsion withparticle sizes ranging from 250-300 nm. To further define the componentsof squalane-Tween 80-pluronic (STP) formulation responsible for CTLinduction, we immunized mice with ova in squalane-Tween 80 (ST) mixture,pluronic-Tween 80 (PT) mixture or squalane-pluronic (SP) mixture and asa control, in squalane (S), Tween 80 (T) or pluronic (P). Mice were alsoimmunized with ova-SAFm (containing 70 μg of MDP) or ova-alum asadjuvant controls. For a positive control, mice were immunized withspleen cells cytoplasmically loaded with soluble ova. Other combinationsand substitutes were also used, and the results are presented in Table1.

For the detection of CTL priming studies, mice were immunized once. Twoweeks after the immunization, spleen cells were mixed with irradiatedEG7-ova (the ova expressing EL4 cells) for five days and tested against⁵¹ Cr-EG7-ova or ⁵¹ Cr-EL4 cells. The results (FIG. 11) demonstrate that30 μg of ova in combination with STP or ST primes class I restricted CTLresponse in mice. The priming of ova specific CTL by ova in STP or byova in ST appears to be better than that induced by spleen cellscytoplasmically loaded with soluble ova. Ova in PT or in SP could induceova specific CTL responses in mice but inconsistently and poorly. UnlikeSAFm, the addition of MDP to ST formulation did not compromise the ovaspecific CTL induction in mice (Table 2). No ova specific CTL inductionoccurred when mice were immunized with ova mixed with the individualcomponents, S, P or T nor when mice were immunized with ova-SAFm orova-alum. Mice immunized with as much as 1 mg ova in (a) HBSS, in (b)SAFm or (c) absorbed to alum did not prime ova specific CTL.

                                      TABLE 2                                     __________________________________________________________________________    Induction of ova specific CTL response is not blocked by ST + MDP                               % cytotoxicity in mice immunized with*                                             ova-ST-MDP                                                                           ova-ST-MDP                                      Stimulator                                                                         Target**                                                                           E-T ova-ST                                                                            ova-ST                                                                             300 μg mouse                                                                      72 μg mouse                                  __________________________________________________________________________    EG7-ova                                                                            EG7-ova                                                                            100:1                                                                             0   100  82     76                                                        33:1                                                                              0   86   67     62                                                        11:1                                                                              0   33   39     25                                                        3:1 0   6    13      3                                                        1:1 0   0     0      0                                                        3:1 0   0     0      0                                              __________________________________________________________________________     *mice were immunized with 30 μg ova in various formulations                **% cytoxicity was calculated by subtracting the percent kill against         antigen nonexpressing cells lines                                        

EXAMPLE 7 Components Necessary for ova Specific Antibody production

Mice were immunized three times at 2 week intervals with 30 μg of ova inHBSS, STP, ST, PT or SP. As a positive control, mice were also immunizedwith ova-SAFm, as SAFm is known to induce a strong antibody response.Seven days after the second and third immunizations, mice were bled andthe sera tested for ova specific antibody response. The results areshown in Table 3. They indicate that mice immunized with ova in STP, STor in SAFm display similar anti-ova responses after two immunizations.

                  TABLE 3                                                         ______________________________________                                        Induction of anti-ova antibody response                                       30 μg ova/animal                                                                      # mice responded/                                                  formulation                                                                              # mice injected                                                                             1/dilution sera titer                                ______________________________________                                        HBSS       0/3           <1/20, <1/20, <1/20                                  STP        3/3           <1/4860, >1/4860, <1/4860                            ST         3/3           >1/4860, >1/4860, >1/4860                            PT         NA            NA, NA, NA                                           SP         NA            NA, NA, NA                                           SAF-M      3/3           1/4860, 1/4860, 1/4860                               ______________________________________                                         *N/A; not available                                                      

EXAMPLE 8 HIVgp120 Specific CTL Induction

HIV gp120 IIIB was used as a second antigen system to determine CTLinduction in STP, ST or in MP-T. Mice were immunized with 1 μg of gp120IIIb in HBSS, STP, PT or in ST. As a control, mice were immunized with 1μg of gp120IIIb in SAFm or CFA (Complete Freund's Adjuvant) or in RIBIadjuvant system containing MPL (monophoshoryl lipid A) and TDM(trehalose dimycolite). Three weeks after the immunization, spleen cellswere prepared and stimulated in vitro with mitomycin treatedtransfectant cells 15-12 or with the 18IIIb peptide. After five days ofculture, the resultant effector cells were tested against vaccinia:gp160IIIB, or parental vaccinia infected P815 cells as targets. The resultsdemonstrate that the gp120-Squalane-TWEEN 80formulation and notgp120-Squalane-TWEEN 80 pluronic formulation or gp120-HBSS induced gp120specific CTL response in mice (Table 4).

                                      TABLE 4                                     __________________________________________________________________________    Induction of gp120 specific CTL response in mice                                                      % cytotoxicity in mice                                                        immunized with*                                       Stimulator                                                                          Target**                                                                            E-T  gp120-HBSS                                                                           gp120-ST                                                                            gp120-STP                                       __________________________________________________________________________    18IIIb/IL2                                                                          vac:gp120                                                                           100:1                                                                              23     42      NA***                                                     33:1 23     38    NA                                                          11:1 0       0    NA                                                           3:1 0      35    NA                                              18IIIb/IL2                                                                          15-12 100:1                                                                              0      50    0                                                           33:1 0      35    0                                                           11:1 0      27    0                                                            3.1 0      18    0                                               18IIIb/IL2                                                                          3T3 + 18IIIb                                                                        100:1                                                                              0      59    13                                                          33:1 0      59    2                                                           11:1 0      57    0                                                            3:1 0      29    0                                               15-12 vac:gp120                                                                           100:1                                                                              35     84    NA                                                          33:1 19     65    NA                                                          11:1 12     37    NA                                                           3:1 0      22    NA                                                           1:1 0       0    NA                                              __________________________________________________________________________     *mice were immunized with 1 μg of gp120III in various formulations         **% cytotoxicity was calculated by subtracting the percent kill against       antigen nonexpressing cell lines                                              ***NA; not available                                                     

EXAMPLE 9 Induction of gp120 Specific Humoral Response in Mice

For the induction of gp120 specific humoral responses, mice wereimmunized with 1 μg of gp120IIIb three times at two-week intervals. Theanimals were bled and tested for the presence of IgG antibodiesdetecting gp120IIIb in a solid phase ELISA assay. The resultsdemonstrate that gp120-ST is a better immunogen than gp120-HBSS,gp120SAFm (Table 5), or gp120-STP.

                  TABLE 5                                                         ______________________________________                                        Induction of anti-gp120 antibody response                                     1 μg gp120/animal                                                                     # mice responded/                                                  formulation                                                                              # mice injected                                                                             1/dilution sera titer                                ______________________________________                                        HBSS       0/3           <1/20, <1/20, <1/20                                  STP        1/3           <1/20, >1/4860, <1/20                                ST         3/3           >1/4860, >1/4860, >1/4860                            PT         3/3           >1/4860, >1/4860, >1/4860                            SP         2/3           <1/20, 1/540, 1/540                                  Saf-M      2/3           1/180, >1/4860, 1/540                                ______________________________________                                    

EXAMPLE 10 gp120 Specific antibody Responses in monkeys

Monkeys (two per group) were immunized with gp120-SAFm, gp120-SPT,gp120-ST, or gp120-HBSS. As a control, a group of monkeys were immunizedwith recombinant vaccinia containing gp160 IIIb. Monkeys were immunizedat two week intervals and bled two weeks and three weeks after thesecond immunization. Pre- and immune sera from each monkey was seriallydiluted and assayed for anti-gp120 activity in an ELISA as described inthe materials and methods. The data (FIG. 12) indicate that monkeysimmunized with gp120-STP or gp120-SAFm induced similar responses inmonkeys. One monkey immunized with gp120-ST, induced anti-gp120 responsesimilar to the gp120-SAFm or gp120-SPT immunized group. One monkeyimmunized with gp120-ST did not induce a strong anti-gp120 responseafter two immunizations.

EXAMPLE 11 In vivo activity of AF in combination with HPV 16 E7

1. Generation of recombinant HPV 16 E7 Protein for Immunization

a) PCR and cloning of the E7 gene

The HPV 16 E7 gene was cloned from a plasmid obtained from Dr. KarenVousden (Ludwig Institute) encoding the E7 gene derived from thecarcinoma cell line CaSki. The coding regions were amplified by PCRusing primers that encode the 5' and 3' ends of the genes flanked by BamHI and Sal I cloning sites. The E7 PCR product was ligated into thepGEX-4T-1 expression vector (Pharmacia Biotech) resulting in the pGEX.E7expression plasmid. E. coli strain XL1-blue (stratagene) was transfectedwith the pGEX.E7 expression plasmid. The sequence of the E7 was obtainedfrom the plasmids of the resulting colonies and was identical to the E7sequence obtained from CaSki cells.

b) Production of purification of bacterially-expressed E7

The pGEX.E7 bacterial expression plasmid encodes aglutathione-S-transferase (GST) fusion protein consisting of the GST atthe amino-terminus, a thrombin protease cleavage site and the E7 proteinat the carboxy-terminus. E7 protein was produced and purified asdescribed in the product information literature from the manufacturer ofthe pGEX-4T-1 vector (Pharmacia Biotech). Briefly, bacteria containingthe pGEX.E7 expression plasmid was induced to express the fusion proteinby the addition of isopropyl b-D-thiogalactosidase to the culturemedium. The cells were harvested and lysed by mild sonication. Thelysate was applied to Glutathione Sepharose 4B (Pharmacia Biotech).After the fusion protein bound to the matrix, the resin was washed toremove non-specifically bound proteins. The bound fusion protein wasdigested with thrombin to release the E7 protein from the GST fusionpartner.

The E7 protein preparation was analyzed by SDS-PAGE and the E7 proteinconcentration was determined by Bradford analysis (BioRad). 9 mg solubleE7 protein was obtained per liter of bacterial culture.

2. Generation of the X21 E7 Transfectant

Coding sequences for the HPV16 E7 protein (see above) have been insertedinto the IDEC proprietary eukaryotic expression plasmid INPEP4. Withinthis vector, E7 expression is controlled by the Cytomegaloviruspromoter/enhancer transcriptional elements. In addition, the first threenucleotides of the E7 coding sequence have been removed and replacedwith an immunoglobulin light chain leader sequence placed immediatelyupstream and in frame with the E7 coding region. Following transfectioninto the mouse cell line X21 individual G418 resistant clones wereexamined by northern blot analyses for E7 message production. Everyclone displayed detectable E7 message. Western blot analysis of celllysates from the two of those clones, 4E7 and 1C7, (HOPE1 and HOPE2respectively) were then performed and demonstrated E7 proteinproduction.

3. In vivo Activity of E7/AF Soluble Antigen Immunization

Female mice of C3H background (H2^(k/k), Harlan Sprague Dawley) wereused in these studies. Animals were maintained according to "Guide forthe Care and Use of Laboratory Animals" (DHHS Publication No. NIH 86-23,Bethesda, Md.:NIH, 1985), and received food and water ad libitum. The E7transfectant cell line HOPE2 H2^(k/k)) was used in these studies. Thetumor cell line was maintained by serial passage in vitro.

This cell line has been shown to maintain E7 cytoplasmic antigenexpression, as detected by western blot analysis, following repeated invitro passages. Tumors were initiated in syngeneic C3H mice bysubcutaneous injection of 150,000 in vitro passaged cells.

Tumors were measured in 2 perpendicular directions at biweeklyintervals. Tumor volume (V) was calculated according to the followingformula:

    V(mm.sup.3)=(L×W.sup.2) divided by 2

where:

L=longest axis measurement in mm

W=perpendicular axis (mm)

Data in Table 6 are presented as tumor Mice (number of tumor bearinganimals over the total number of animals injected). Data in FIGS. 13 and14 are presented as median tumor size (mm³) of each treatment or controlgroup. Each treatment group was compared to a control group that did notreceive therapy. Therapy began 10 days after incoculation of HOPE2cells, when a majority of the tumors were palpable (approx. 50-75 mm³).Therapy was initiated by immunization of mice with soluble E7 protein inAF or Alum adjuvants (subcutaneously in a total volume of 0.2 ml).Directly before immunization, AF was mixed for 60 seconds with E7protein in Hanks Balanced Salt Solution (HBSS) such that each mousereceived either 30 ug or 90 ug E7 protein 0.2 ml. Alum (Pierce ChemicalCo.) was mixed with E7 protein, according to instructions by themanufacture, such that each animal received 90 ug E7 protein in 0.2 mlper mouse. Animals in a second treatment group received a secondimmunization 9 days later (19 days after tumor cell inoculation).Booster Immunization were prepared immediately before inoculation, asdescribed above.

In this example (Table 6: Xp #233), 41 days after tumor cell inoculationonly 4/8 and 5/8 of mice receiving a single injection of soluble E7 inaF (30 ug or 90 ug respectively) had measurable tumors. In contrast, allof the mice immunized with E7 protein in Alum (8/8) had actively growingtumors. Additionally, as shown in FIG. 13, significant inhibition oftumor growth was observed only in treatment groups immunized with E7protein in aF as compared to control (untreated) or Alum treatmentgroups. Inhibition of tumor growth (FIG. 13) or increased tumorregression rates (Table 6 was not observed in mice that received asingle injection of E7 in Alum.

Similar results were also observed using treatment groups that receivedtwo immunizations at days 10 and 19 after tumor challenge (Table 6 andFIG. 14), although some tumor growth retardation was observed with micereceiving two injections of E7 in Alum.

The results indicate that significant antitumor activity as measured bya decreased number of tumor bearing mice and inhibition of tumor growthwas observed following immunization of soluble E7 in AF. In contrast,all animals immunized with either a single or double injection ofsoluble E7 protein in Alum had growing tumors. In summary, immunizationwith soluble E7 protein in AF resulted in a significant inhibition oftumor cell growth that was not observed using soluble E7 immunization inAlum.

                  TABLE 6                                                         ______________________________________                                        Antitumor activity of soluble E7 immunization in adjuvant                     Exp. # Treatment  Dose (ug/mouse)                                                                           Tumor Animals.sup.a Day 41                      ______________________________________                                        223    Control    --          7/8                                             223    E7 in AF   30 ug × 1.sup.b                                                                     4/8                                             223    E7 in AF   90 ug × 1                                                                           5/8                                             223    E7 in Alum 90 ug × 1                                                                           8/8                                             223    E7 in AF   30 ug × 2.sup.c                                                                     3/8                                             223    E7 in AF   90 ug × 2                                                                           1/4                                             223    E7 in Alum 90 ug × 2                                                                           8/8                                             ______________________________________                                         .sup.a Number of tumor bearing mice/total number inoculated                   .sup.b All immunizations started on Day 10 post implant                       .sup.c Second immunization (×2) on Day 19 post implant             

Other embodiments are within the following claims.

We claim:
 1. A composition comprising an antigen mixed with amicrofluidized antigen formulation comprising:(a) a stabilizingdetergent, (b) a micelle-forming agent, and (c) a biodegradable andbiocompatible oil, said antigen formulation being formulated as a stableoil-in-water emulsion, said antigen formulation being substantially freeof immunostimulating peptides and wherein said composition uponadministration to an animal selected from the group consisting ofhumans, domesticated animals and agricultural animals is capable ofinducing a specific cytotoxic T-lymphocyte response against the antigencontained in the composition.
 2. The composition of claim 1, whereinsaid antigen is chosen from antigenic portions of the HIV antigens:gp160, gag, pol, Nef, Tat, and Rev; the malaria antigens: CS protein andSporozoite surface protein 2; the Hepatitis B surface antigens: Pre-S1,Pre-S2, HBc Ag, and HBe Ag; the influenza antigens: HA, NP and NA;Hepatitis A surface antigens; the Herpes virus antigens: EBV gp340, EBVgp85, HSV gB, HSV gD, HSV gH, HSV early protein product, cytomegalovirusgB, cytomegalovirus gH, and IE protein gp72; the respiratory syncytialvirus antigens: F protein, G protein, and N protein; and the tumorantigens: carcinoma CEA, carcinoma associated mucin, carcinoma P21,carcinoma P53, melanomaMPG, melanoma p97, carcinoma Neu oncogeneproduct, carcinoma p53 gene product, and mutated p21 ras protein.
 3. Acomposition comprising an antigen mixed with a microfluidized antigenformulation comprising:(a) a stabilizing detergent, (b) amicelle-forming agent, and (c) a biodegradable and biocompatible oil,said antigen formulation being formulated as a stable oil-in-wateremulsion, said antigen formulation lacking immunostimulating peptidesand wherein said composition upon administration to an animal selectedfrom the group consisting of humans, domesticated animals andagricultural animals is capable of inducing a specific cytotoxicT-lymphocyte response against the antigen contained in the composition.4. The composition of claim 3, wherein said antigen formulation consistsessentially of said detergent, agent, and oil.
 5. The composition ofclaim 3, wherein said antigen formulation is non-toxic to said human ordomesticated or agricultural animal.
 6. The composition of claim 3,wherein said antigen is chosen from the HIV antigens: gp160, gag, pol,Nef, Tat, and Rev; the malaria antigens: CS protein and Sporozoitesurface protein 2; the Hepatitis B surface antigens: Pre-S1, Pre-S2, HBcAg, and HBe Ag; the influenza antigens: HA, NP and NA; Hepatitis Asurface antigens; the Herpes virus antigens: EBV gp340, EBV gp85, HSVgB, HSV gD, HSV gH, HSV early protein product, cytomegalovirus gB,cytomegalovirus gH, and IE protein gP72; the respiratory syncytial virusantigens: F protein, G protein, and N - protein; and the tumor antigens:carcinoma CEA, carcinoma associated mucin, carcinoma P21, carcinoma P53,melanoma MPG, melanoma p97, carcinoma Neu oncogene product, carcinomap53 gene product, a human papillomavirus antigen, the prostate specificantigen (PSA) and mutated p21 ras protein.
 7. The composition of claim 1wherein the stabilizing detergent is selected from the group consistingof polysorbate 80, Tween 20, Tween 40, Tween 60, Zwittergent 3-12,Teepol HB7 and Span
 85. 8. The composition of claim 1 wherein saiddetergent is provided in an amount ranging from approximately 0.05 to0.5%.
 9. The composition of claim 8 wherein said amount of detergent isabout 0.2%.
 10. The composition of claim 1 wherein said micelle-formingagent comprises a hydrophile-lipophile balance of between 0 and
 2. 11.The composition of claim 1 wherein said micelle-forming agent isselected from the group consisting of poloxamer 401, Pluronic L62LF,Pluronic L101, Pluronic L64, PEG1000, Tetronic 1501, Tetronic 150R1,Tetronic 701, Tetronic 901, Tetronic 1301, and Tetronic 130R1.
 12. Thecomposition of claim 1 wherein the amount of said micelle-forming agentranges from between 0.5 to 10%.
 13. The composition of claim 12 whereinthe amount of said micelle-forming agent ranges from between 1.25 and5%.
 14. The composition of claim 1 wherein the oil exhibits a meltingtemperature less than 60° C.
 15. The composition of claim 14 wherein theoil is selected from the group consisting of squalene, squalane,eicosane, tetratetracontane, pristane, glycerol, and vegetable oils. 16.The composition of claim 1 wherein the amount of the oil ranges frombetween 1 and 10%.
 17. The composition of claim 16 wherein the amount ofthe oil ranges from between 2.5 and 5%.
 18. The composition of claim 1which comprises less than 20 micrograms of muramyl dipeptide.
 19. Thecomposition of claim 18 does not comprise any muramyl dipeptide.
 20. Thecomposition of claim 1 wherein the detergent is polysorbate 80, and themicelle-forming agent is poloxamer
 401. 21. The composition of claim 20wherein the oil is squalane.
 22. The composition of claim 1 wherein thedetergent is selected from the group consisting of Tween 20, Tween 40and Tween 80; the oil is selected from the group consisting of squalane,eicosane, and pristane and the micelle-forming agent is selected fromthe group consisting of Pluronic L62LF and polyoxamer
 401. 23. Thecomposition of claim 1 wherein the particle sizes in the compositionrange from 250 to 300 nm.